Liposome having an exchangeable component

ABSTRACT

The present invention provides a fusogenic liposome comprising a lipid capable of adopting a non-lamellar phase, yet capable of assuming a bilayer structure in the presence of a bilayer stabilizing component; and a bilayer stabilizing component reversibly associated with the lipid to stabilize the lipid in a bilayer structure. Such fusogenic liposomes are extremely advantageous because the rate at which they become fusogenic can be not only predetermined, but varied as required over a time scale ranging from minutes to days. Control of liposome fusion can be achieved by modulating the chemical stability and/or exchangeability of the bilayer stabilizing component(s). The fusogenic liposomes of the present invention can be used to deliver drugs, peptide, proteins, RNA, DNA or other bioactive molecules to the target cells of interest.

This application is a continuation of U.S. patent application Ser. No.08/485,608, filed Jun. 7, 1995, now U.S. Pat. No. 5,885,613, whichclaims priority to U.S. application Ser. No. 08/316,407, filed Sep. 30,1994.

BACKGROUND OF THE INVENTION

It is well recognized in the medical field that the most effectiveprocedure for treating localized disease is to direct the pharmaceuticalor drug agent (hereinafter “drugs”) to the affected area, therebyavoiding undesirable toxic effects of systemic treatment. Techniquescurrently being used to deliver drugs to specific target sites withinthe body involve the utilization of time-release capsules or gelmatrices from which drugs slowly “leak,” or the use of implantable“syringes” that mechanically release drugs into muscles or into theblood stream. Another, and perhaps more effective delivery system,encompasses the use of liposomes containing the appropriate drug orchemical. The liposome with encapsulated drug is directed to thespecific area of interest and, thereafter, the drug is released. Thecarrying out of this latter step is the most problematic and, in fact,the greatest barrier to the use of liposomes as drug carriers is makingthe liposomes release the drugs on demand at the target site ofinterest.

Liposomes are vesicles comprised of one or more concentrically orderedlipid bilayers which encapsulate an aqueous phase. They are normally notleaky, but can become leaky if a hole or pore occurs in the membrane, ifthe membrane is dissolved or degrades, or if the membrane temperature isincreased to the phase transition temperature, T_(c). Current methods ofdrug delivery via liposomes require that the liposome carrier willultimately become permeable and release the encapsulated drug at thetarget site. This can be accomplished, for example, in a passive mannerwherein the liposome bilayer degrades over time through the action ofvarious agents in the body. Every liposome composition will have acharacteristic half-life in the circulation or at other sites in thebody and, thus, by controlling the half-life of the liposomecomposition, the rate at which the bilayer degrades can be somewhatregulated.

In contrast to passive drug release, active drug release involves usingan agent to induce a permeability change in the liposome vesicle.Liposome membranes can be constructed so that they become destabilizedwhen the environment becomes acidic near the liposome membrane (see,e.g., Proc. Natl. Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908(1989)). When liposomes are endocytosed by a target cell, for example,they can be routed to acidic endosomes which will destabilize theliposome and result in drug release. Alternatively, the liposomemembrane can be chemically modified such that an enzyme is placed as acoating on the membrane which slowly destabilizes the liposome. Sincecontrol of drug release depends on the concentration of enzyme initiallyplaced in the membrane, there is no real effective way to modulate oralter drug release to achieve “on demand” drug delivery. The sameproblem exists for pH-sensitive liposomes in that as soon as theliposome vesicle comes into contact with a target cell, it will beengulfed and a drop in pH will lead to drug release.

In addition to the foregoing methods, a liposome having a predeterminedphase transition temperature, T_(c), above body temperature can be usedto achieve active drug delivery. In this method, the body temperaturewill maintain the liposome below the T_(c) so that the liposome will notbecome leaky when placed in the body. This method of drug release iscapable of “on demand” drug delivery since such liposomes experience agreatly increased membrane permeability at their T_(c) which, in turn,enables drug or chemical release. To release drugs from such phasetransition liposomes when in the body, heat must be applied until theT_(c) is achieved. Unfortunately, the application of heat can, initself, create problems within the body and, frequently, the adverseeffects of the heat treatment outweigh the beneficial effects of usingthe liposome as a drug delivery vehicle. Moreover, such liposomes mustbe made of highly purified and expensive phase transition temperaturephospholipid materials.

In view of the foregoing, there exists a need in the art for a methodfor targeted drug delivery that overcomes the disadvantages of thecurrently available methods. Specifically, a parenteral delivery systemis required that would be stable in the circulation, followingintravenous administration, allowing retention of encapsulated orassociated drug or therapeutic agent(s). This delivery system would becapable of accumulating at a target organ, tissue or cell via eitheractive targeting (e.g., by incorporating an antibody or hormone on thesurface of the liposomal vehicle) or via passive targeting, as seen forlong-circulating liposomes. Following accumulation at the target site,the liposomal carrier would become fusogenic, without the need for anyexternal stimulus, and would subsequently release any encapsulated orassociated drug or therapeutic agent in the vicinity of the target cell,or fuse with the target cell plasma membrane introducing the drug ortherapeutic agent into the cell cytoplasm. In certain instances, fusionof the liposomal carrier with the plasma membrane would be preferredbecause this would provide more specific drug delivery and, hence,minimize any adverse effects on normal, healthy cells or tissues. Inaddition, in the case of therapeutic agents such as DNA, RNA, proteins,peptides, etc., which are generally not permeable to the cell membrane,such a fusogenic carrier would provide a mechanism whereby thetherapeutic agent could be delivered to its required intracellular siteof action. Further, by avoiding the endocytic pathway, the therapeuticagent would not be exposed to acidic conditions and/or degradativeenzymes that could inactivate said therapeutic agent. Quitesurprisingly, the present invention addresses this need by providingsuch a method.

SUMMARY OF THE INVENTION

The present invention provides a fusogenic liposome comprising a lipidcapable of adopting a non-lamellar phase, yet capable of assuming abilayer structure in the presence of a bilayer stabilizing component;and a bilayer stabilizing component reversibly associated with the lipidto stabilize the lipid in a bilayer structure. Such fusogenic liposomesare extremely advantageous because the rate at which they becomefusogenic can be not only predetermined, but varied as required over atime scale ranging from minutes to days. Control of liposome fusion canbe achieved by modulating the chemical stability and/or exchangeabilityof the bilayer stabilizing component(s).

Lipids which can be used to form the fusogenic liposomes of the presentinvention are those which adopt a non-lamellar phase under physiologicalconditions or under specific physiological conditions, e.g., in thepresence of calcium ions, but which are capable of assuming a bilayerstructure in the presence of a bilayer stabilizing component. Lipidswhich adopt a non-lamellar phase include, but are not limited to,phosphatidylenthanolamines, ceramides, glycolipids, or mixtures thereof.Such lipids can be stabilized in a bilayer structure by bilayerstabilizing components which are either bilayer forming themselves, orwhich are of a complementary dynamic molecular shape. More particularly,the bilayer stabilizing components of the present invention must becapable of stabilizing the lipid in a bilayer structure, yet they mustbe capable of exchanging out of the liposome, or of being chemicallymodified by endogenous systems so that, with time, they lose theirability to stabilize the lipid in a bilayer structure, thereby allowingthe liposome to become fusogenic. Only when liposomal stability is lostor decreased can fusion of the liposome with the plasma membrane of thetarget cell occur.

By controlling the composition and concentration of the bilayerstabilizing component, one can control the chemical stability of thebilayer stabilizing component and/or the rate at which the bilayerstabilizing component exchanges out of the liposome and, in turn, therate at which the liposome becomes fusogenic. In addition, othervariables including, for example, Ph, temperature, ionic strength, etc.can be used to vary and/or control the rate at which the liposomebecomes fusogenic.

The fusogenic liposomes of the present invention are ideally suited to anumber of therapeutic, research and commercial applications. Intherapeutic applications, for example, the initial stability of thefusogenic liposome would allow time for the liposome to achieve accessto target organs or cells before attaining its fusogenic state, therebyreducing non-specific fusion immediately following administration.

In addition, the fusogenic liposomes of the present invention can beused to deliver drugs, peptide, proteins, RNA, DNA or other bioactivemolecules to the target cells of interest. In this embodiment, thecompound or molecule to be delivered to the target cell can beencapsulated in the aqueous interior of the fusogenic liposome andsubsequently introduced into the cytoplasma (initially) upon fusion ofthe liposome with the cell plasma membrane. Alternatively, molecules orcompounds can be embedded within the liposome bilayer and, in this case,they would be incorporated into the target cell plasma membrane uponfusion.

As such, in another embodiment, the present invention provides a methodfor delivering a therapeutic compound to a target cell at apredetermined rate, the method comprising: administering to a hostcontaining the target cell a fusogenic liposome which comprises abilayer stabilizing component, a lipid capable of adopting anon-lamellar phase, yet capable of assuming a bilayer structure in thepresence of the bilayer stabilizing component, and a therapeuticcompound or a pharmaceutically acceptable salt thereof. Administrationmay be by a variety of routes, but the therapeutic compounds arepreferably given intravenously or parenterally. The fusogenic liposomesadministered to the host may be unilamellar, having a mean diameter of0.05 to 0.45 microns, more preferably from 0.05 to 0.2 microns.

In a final embodiment, the present provides a method of stabilizing in abilayer structure a lipid which is capable of adopting a non-lamellarphase by combining the lipid(s) with a bilayer stabilizing component.Once stabilized, the lipid mixture can be used to form the fusogenicliposomes of the present invention. The bilayer stabilizing component isselected, however, to be exchangeable such that upon loss of thiscomponent from the liposome, the liposome is destabilized and becomesfusogenic.

Other features, objects and advantages of the invention and itspreferred embodiments will become apparent from the detailed descriptionwhich follows.

BRIEF DESCRIPTION OF THE DRAWINGS

There is at least one color drawing in this application.

FIG. 1 illustrates the concentration dependence of bilayer stabilizationby a bilayer stabilizing component (BSC). Multilamellar vesicles wereprepared, as described in the examples, from mixtures ofDOPE:cholesterol:DOPE-PEG₂₀₀₀, 1:1:N, where N is the proportion ofDOPE-PEG₂₀₀₀ as indicated in the FIG. 1. ³¹P-NMR spectra were determinedat 20° C. after the sample had been allowed to equilibrate for 30minutes.

FIG. 2 illustrates the temperature dependence of bilayer stabilizationby BSC. Multilamellar vesicles were prepared, as described in theexamples, from mixtures of DOPE:cholesterol:DOPE-PEG₂₀₀₀ at a ratio of:A, 1:1:0.1; or B, 1:1:0.25. The samples were cooled to 0° C. and ³¹p-NMRspectra were determined from 0° C. to 60° C. at 10° C. intervals. Thesamples were allowed to equilibrate at each temperature for 30 min.prior to data accumulation.

FIG. 3 illustrates the effect of headgroup size on the bilayerstabilizing ability of BSC. Multilamellar vesicles were prepared fromeither A, DOPE:cholesterol: DOPE-PEG₂₀₀₀, 1:1:0.05, or B,DOPE:cholesterol:DOPE-PEG₅₀₀₀, 1:1:0.05. Other conditions were the sameas for FIG. 2.

FIG. 4 illustrates the effect of the acyl chain composition on thebilayer stabilizing ability of BSC. Multilamellar vesicles wereprepared, as described in the examples, from either A,DOPE:cholesterol:DMPE-PEG₂₀₀₀, 1:1:0.1, B,DOPE:cholesterol:DPPE-PEG₂₀₀₀, 1:1:0.1, or C,DOPE:cholesterol:DSPE-PEG₂₀₀₀, 1:1:0.1. Other conditions were the sameas for FIG. 2.

FIG. 5 illustrates the ability of PEG-Ceramide to act as a bilayerstabilizing component. Multilamellar vesicles were prepared, asdescribed in the examples, from DOPE:cholesterol:egg ceramide-PEG₂₀₀₀ ata ratio of A, 1:1:0.1 or B, 1:1:0.25. Other conditions were the same asfor FIG. 2.

FIG. 6 illustrates the freeze-fracture electron micrograph of MLVsprepared from DOPE:cholesterol:DOPE-PEG₂₀₀₀ (1:1:0.1). The samples wereprepared as described in the examples. The bar represents 500 nm.

FIG. 7 illustrates the freeze-fracture electron micrograph of LUVsprepared from DOPE:cholesterol:DOPE-PEG₂₀₀₀ (1:1:0.1). The samples wereprepared as described in the examples. The bar represents 500nm.

FIG. 8 illustrates the elution profiles of LUVs prepared fromDOPE:cholesterol:DSPE-PEG₂₀₀₀, and micelles composed of DSPE-PEG₂₀₀₀.LUVs were prepared, as described in the examples, fromDOPE:cholesterol:DSPE-PEG₂₀₀₀ (1:1:0.1) with trace amounts of ¹⁴C-DPPC(Δ) and ³H-DSPE-PEG₂₀₀₀. () They were chromatographed as described inthe examples. In a separate experiment, micelles were prepared fromDSPE-PEG₂₀₀₀ labelled with ³H-DSPE-PEG₂₀₀₀ (◯) and chromatographed onthe same Sepharose 4B column.

FIG. 9 illustrates the inhibition of fusion by PEG-PE. Liposomes wereprepared from equimolar mixtures of DOPE and POPS containing (a) 0; (b)0.5; (c) 1 or (d) 2 mol % DMPE-PEG₂₀₀₀. In addition to the above lipids,labelled liposomes also contained the fluorescent lipids NBD-PE andRh-PE at 0.5 mol %. Fluorescently labelled liposomes (finalconcentration 60 1μM) were incubated at 37° C. for 30s before theaddition of a three-fold excess of unlabelled liposomes, followed oneminute later by CaCl₂ (final concentration 5 mM).

FIG. 10 illustrates the recovery of fusogenic activity after PEG-PEremoval. Fusion between fluorescently labelled and unlabelled liposomescontaining 2 mol % DMPE-PEG₂₀₀₀ was assayed as described under FIG. 9,except that one minute after addition of CaCl₂, a 12-fold excess (overlabelled vesicles) of POPC liposomes (curve a) or an equivalent volumeof buffer (curve b) was added.

FIG. 11 illustrates the concentration dependence of recovery offusogenic activity. Fusion between fluorescently labelled and unlabelledliposomes containing (a) 2; (b) 3; (c) 5 or (d) 10 mol % DMPE-PEG₂₀₀₀was assayed as described under FIG. 10, except that POPC liposomes wereadded as a 36fold excess over labelled vesicles.

FIGS. 12A and B illustrate programmable fusion. Fusion betweenfluorescently labelled and unlabelled liposomes containing 2 mol % ofthe indicated PE-PEG₂₀₀₀ was assayed as described under FIG. 10. The %fusion was calculated as described in the examples. (A) DMPP-PEG₂₀₀₀(); DPPE-PEG₂₀₀₀ (♦); DSPE-PEG₂₀₀₀ (▾); and (B) DOPE-PEG₂₀₀₀ (▾), eggceramide-PEG₂₀₀₀ (▴).

FIGS. 13 A and B illustrate the effect of PEG molecular weight onfusion. (A) Assays were carried out as described in FIG. 9 usingliposomes which contained (a) 0; (b) 0.25; (c) 0.5 or (d) 1 mol %DMPE-PEG₅₀₀₀; and (B) Assays were performed as described under FIG. 12using liposomes which contained 1 mol % DMPE-PEG₅₀₀₀ (); DPPE-PEG₅₀₀₀(♦) or DSPE-PEG₅₀₀₀ (▾).

FIG. 14 illustrates the comparison of PEG₂₀₀₀ to PEG₅₀₀₀ at equalconcentration of oxyethylene groups. Liposomes contained either 2 mol %PEG₅₀₀₀ (upper curve) or 5 mol % PEG₂₀₀₀ (lower curve). Other conditionswere as described under FIG. 11.

FIG. 15 illustrates the effect of salt concentration on fusion ofDOPE:DODAC Liposomes. Liposomes were prepared from DOPE:DODAC (85:15).Donor liposomes also contained the fluorescent lipids, NBD-PE and Rh-PEat 0.5 mol %. Donor liposomes (final concentration 60 μM) were incubatedat 37° C. for 30s before the addition of a three-fold excess ofunlabelled acceptor liposomes followed 1 min later by NaCl to give theindicated final concentration.

FIG. 16 illustrates the inhibition of fusion of DOPE:DODAC liposomes byPEG-PE. Liposomes were prepared from either DOPE:DODAC (85:15) orDOPE:DODAC:DMPE-PEG₂₀₀₀ (83:15:2). Fusion was assayed as described underFIG. 1 using 300 mM NaCl.

FIG. 17 illustrates the recovery fusogenic activity after PEG removal.Liposomes were prepared from either DOPE:DODAC:ceramide(C8:0)-PEG₂₀₀₀,83:15:2 or DOPE:cholesterol:ceramide(C8:0)-PEG₂₀₀₀, 38:45:15:2. Fusionwas assayed as described under FIG. 2 except that at the indicated timesa 30 fold excess (over donors) of liposomes composed of POPC orPOPC:cholesterol (55:45) was added.

FIG. 18 illustrates the effect of the lipid anchor on the rate ofPEG-lipid removal. Fluorescently labelled and unlabelled liposomes wereprepared from DOPE:DODAC:PEG-lipid, 83:15:2, using DMPE-PEG₂₀₀₀ (),ceramide(egg)-PEG₂₀₀₀ or (C14:0) ceramide-PEG₂₀₀₀ (♦). Labelledliposomes were mixed with a 3 fold excess of unlabelled liposomes and300 mM NaCl in a cuvette in a dark water bath at 37° C. At zero time a13-fold excess (over labelled vesicles) of POPC liposomes was added andthe fluorescence intensity was measured at the indicated times. At theend of the assay Triton X-100 (0.5% final) was added to eliminate energytransfer and the % fusion was calculated from the change in efficiencyof energy transfer. Maximum fusion was calculated from a standard curveof energy transfer efficiency against the molar fraction of Rh-PE in themembrane assuming complete mixing of labelled and unlabelled liposomes.

FIG. 19 illustrates the inhibition of fusion betweenDOPE:cholesterol:DODAC liposomes and anionic liposomes by PEG-ceramide.Liposomes were prepared from DOPE:cholesterol:DODAC, 40:45:15 (no PEG)or DOPE:cholesterol:DODAC:(C14:0) ceramide-PEG₂₀₀₀, 36:45:15:4 (4% PEG).Acceptor liposomes were prepared from DOPE:cholesterol:POPS, 25:45:30. Athree-fold excess of acceptors was added to labelled vesicles after 30sand the fluorescence monitored at 517 nm with excitation at 465 nm.

FIG. 20 illustrates the recovery of fusogenic activity upon PEG removal.Donor liposomes (50 μM) were prepared from DOPE:cholesterol:DODAC:(C14:0)ceramide-PEG₂₀₀₀, 36:45:15:4 and mixed with acceptor liposomes(150 μM) prepared from DOPE:cholesterol:POPS, 25:45:30. At zero timeeither 1 mM POPC:cholesterol liposomes (▾) or an equivalent volume ofbuffer () was added. Fluorescence was monitored at 517 nm withexcitation at 465nm.

FIG. 21 illustrates the inhibition of fusion betweenDOPE:cholesterol:DODAC liposomes and erythrocyte ghosts by,PEG-ceramide. Liposomes were prepared from DOPE:cholesterol:DODAC,40:45:15 (no PEG) or DOPE:cholesterol:DODAC:(C_(14:0))ceramide-PEG₂₀₀₀ ,36:45:15:4 (4% PEG). Ghosts (50 μM phospholipid) were added to donors(50 μM total lipid) after 30s and the fluorescence monitored at 517 nmwith excitation at 465 nm.

FIGS. 22A-E illustrate the fusion of fluorescent liposomes composed ofDOPE:cholesterol:DODAC (40:45:15) or DOPE:cholesterol:DODAC:PEG-ceramide(35:45:15:5). LUVs composed of DOPE:cholesterol:DODAC (40:45:15) fusedwith RBCs (panels a and b); incorporation of PEG-ceramide (C8:0) intothe LUVs at 5 mol % blocked fusion (panels c and d); however, when anexogenous sink for the PEG-ceramide was included, fusogenic activity wasrecovered within minutes (panels e and f). Panels a,c and e are viewsunder phase contrast, and panels b,d and f are the same fields viewunder fluorescent light.

FIGS. 23E-F illustrate the results when PEG-ceramides with longer fattyamide chains (C14:0) are used and the liposomes are pre-incubation withan exogenous sink prior to the addition of the RBCs. No fusion wasobserved after pre-incubation of the fusogenic LUVs with the sink forfive minutes prior to addition of RBC (panels a and b); after a 1 hourpre-incubation, some fusion with RBCs was observed (panels c and d);however, with longer incubations times (2 hours), the pattern offluorescent labeling changed and extensive punctate fluorescence wasobserved (panels e and f). Panels a,c and e are views under phasecontrast, and panels b,d and f are the same fields view underfluorescent light.

FIGS. 24A-F illustrate the results when PEG-ceramides with longer fattyamide chains (C20:0) are used and the liposomes are preincubation withan exogenous sink prior to the addition of the RBCs. No fusion wasobserved after pre-incubation of the LUVs with the sink for five minutes(panels a and b), 1 hour (panels c and d) or 2 hours (panels e and f).Panels a, c and e are views under phase contrast, and panels b,d and fare the same fields view under fluorescent light.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In one embodiment of the present invention, a fusogenic liposome isprovided, the fusogenic liposome comprising: a lipid capable of adoptinga non-lamellar phase, yet capable of assuming a bilayer structure in thepresence of a bilayer stabilizing component; and a bilayer stabilizingcomponent reversibly associated with the lipid to stabilize the lipid ina bilayer structure. Such fusogenic liposomes are advantageous becausethe rate at which they become fusogenic can be not only predetermined,but varied as required over a time scale of a few minutes to severaltens of hours. It has been found, for example, that by controlling thecomposition and concentration of the bilayer stabilizing component, onecan control the rate at which the bilayer stabilizing componentexchanges out of the liposome and, in turn, the rate at which theliposome becomes fusogenic.

The polymorphic behavior of lipids in organized assemblies can beexplained qualitatively in terms of the dynamic molecular shape concept(see, Cullis et al., in “Membrane Fusion” (Wilschut, J. and D. Hoekstra(eds.), Marcel Dekker, Inc., New York, (1991)). When the effectivecross-sectional areas of the polar head group and the hydrophobic regionburied within the membrane are similar then the lipids have acylindrical shape and tend to adopt a bilayer conformation. Cone-shapedlipids which have polar head groups that are small relative to thehydrophobic component, such as unsaturated phosphatidylethanolamines,prefer non-bilayer phases such as inverted micelles or inverse hexagonalphase (H_(II)). Lipids with head groups that are large relative to theirhydrophobic domain, such as lysophospholipids, have an inverted coneshape and tend to form micelles in aqueous solution. The phasepreference of a mixed lipid system depends, therefore, on thecontributions of all the components to the net dynamic molecular shape.As such, a combination of cone-shaped and inverted cone-shaped lipidscan adopt a bilayer conformation under conditions where either lipid inisolation cannot (see, Madden and Cullis, Biochim. Biophys. Acta,684:149-153 (1982)).

A more formalized model is based on the intrinsic curvature hypothesis(see, e.g., Kirk, et al., Biochemistry, 23:1093-1102 (1984)). This modelexplains phospholipid polymorphism in terms of two opposing forces. Thenatural tendency of a lipid monolayer to curl and adopt its intrinsic orequilibrium radius of curvature (R_(o)) which results in an elasticallyrelaxed monolayer is opposed by the hydrocarbon packing constraints thatresult. Factors that decrease the intrinsic radius of curvature, such asincreased volume occupied by the hydrocarbon chains when double bondsare introduced, tend to promote H_(II) phase formation. Conversely, anincrease in the size of the headgroup increases R_(o) and promotesbilayer formation or stabilization. Introduction of apolar lipids thatcan fill the voids between inverted lipid cylinders also promotes H_(II)phase formation (see, Gruner, et al., Proc. Natl. Acad. Sci. USA,82:3665-3669 (1989); Sjoland, et al., Biochemistry, 28:1323-1329(1989)).

Lipids which can be used to form the fusogenic liposomes of the presentinvention are those which adopt a non-lamellar phase under physiologicalconditions or under specific physiological conditions, e.g., in thepresence of calcium ions, but which are capable of assuming a bilayerstructure in the presence of a bilayer stabilizing component. Suchlipids include, but are not limited to, phosphatidylenthanolamines,ceramides, glycolipids, or mixtures thereof. Other lipids known to thoseof skill in the art to adopt a non-lamellar phase under physiologicalconditions can also be used. Moreover, it will be readily apparent tothose of skill in the art that other lipids can be induced to adopt anon-lamellar phase by various non-physiological changes including, forexample, changes in pH or ion concentration (e.g., in the presence ofcalcium ions) and, thus, they can also be used to form the fusogenicliposomes of the present invention. In a presently preferred embodiment,the fusogenic liposome is prepared from a phosphatidylethanolamine. Thephosphatidylethanolamine can be saturated or unsaturated. In a presentlypreferred embodiment, the phosphatidylyethanolamine is unsaturated. Inan equally preferred embodiment, the fusogenic liposome is prepared froma mixture of a phosphatidylethanolamine (saturated or unsaturated) and aphosphatidylserine. In another equally preferred embodiment, thefusogenic liposome is prepared from a mixture of aphosphatidylethanolamine (saturated or unsaturated) and a cationiclipid.

Examples of suitable cationic lipids include, but are not limited to,the following: DC-Chol,3β-(N-(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (see, Gao, etal., Biochem. Biophys. Res. Comm 179:280-285 (1991); DDAB,N,N-distearyl-N,N-dimethylammonium bromide; DMRIE,N-(1,2dimyristyloxypro-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide; DODAC, N,N-dioleyl-N,N-dimethylammonium chloride (see, commonlyowned U.S. patent application Ser. No. 08/316,399, filed Sep. 30, 1994,which is incorporated herein by reference); DOGS,diheptadecylamidoglycyl spermidine; DOSPA,N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate; DOTAP,N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; andDOTMA, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride. Ina presently preferred embodiment, N,N-dioleoyl-N,N-dimethylammoniumchloride is used in combination with a phosphatidylethanolamine.

In accordance with the present invention, lipids adopting a non-lamellarphase under physiological conditions can be stabilized in a bilayerstructure by bilayer stabilizing components which are either bilayerforming themselves, or which are of a complementary dynamic shape. Thenon-bilayer forming lipid is stabilized in the bilayer structure onlywhen it is associated with, i.e., in the presence of, the bilayerstabilizing component. In selecting an appropriate bilayer stabilizingcomponent, it is imperative that the bilayer stabilizing component becapable of transferring out of the liposome, or of being chemicallymodified by endogenous systems such that, with time, it loses itsability to stabilize the lipid in a bilayer structure. Only whenliposomal stability is lost or decreased can fusion of the liposome withthe plasma membrane of the target cell occur. The bilayer stabilizingcomponent is, therefore, “reversibly associated” with the lipid and onlywhen it is associated with the lipid is the lipid constrained to adoptthe bilayer structure under conditions where it would otherwise adopt anon-lamellar phase. As such, the bilayer stabilizing components of thepresent invention must be capable of stabilizing the lipid in a bilayerstructure, yet they must be capable of exchanging out of the liposome,or of being chemically modified by endogenous systems so that, withtime, they lose their ability to stabilize the lipid in a bilayerstructure, thereby allowing the liposome to become fusogenic.

Examples of suitable bilayer stabilizing components include, but are notlimited to, lipid, lipid-derivatives, detergents, proteins and peptides.In a presently preferred embodiment, the bilayer stabilizing componentis polyethyleneglycol conjugated to, i.e., coupled to, aphosphatidylethanolamine. In an equally preferred embodiment, thebilayer stabilizing component is polyethyleneglycol conjugated to aceramide. Polyethyleneglycol can be conjugated to aphosphatidylethanolamine or, alternatively, to a ceramide using standardcoupling reactions known to and used by those of skill in the art. Inaddition, preformed polyethyleneglycol-phosphatidylethanolamineconjugates are commercially available from Avanti Polar Lipids(Alabaster, Ala.).

Polyethyleneglycols of varying molecular weights can be used to form thebilayer stabilizing components of the present invention.Polyethyleneglycols of varying molecular weights are commerciallyavailable from a number of different sources or, alternatively, they canbe synthesized using standard polymerization techniques well-known tothose of skill in the art. In a presently preferred embodiment, thepolyethylene glycol has a molecular weight ranging from about 200 toabout 10,000, more preferably from about 1,000 to about 8,000, and evenmore preferably from about 2,000 to about 6,000. Generally, it has beenfound that increasing the molecular weight of the polyethyleneglycolreduces the concentration of the bilayer stabilizing component requiredto achieve stabilization.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated topolyethyleneglycol to form the bilayer stabilizing component. Suchphosphatidylethanolamines are commercially available, or can be isolatedor synthesized using conventional techniques known to those of skill inthe art. Phosphatidylethanolamines containing saturated or unsaturatedfatty acids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, the following: dimyristoylphosphatidylethanolamine (DMPE),dipalmitoylphosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE) anddistearoylphosphatidyl-ethanolamine (DSPE).

As with the phosphatidylethanolamines, ceramides having a variety ofacyl chain groups of varying chain lengths and degrees of saturation canbe coupled to polyethyleneglycol to form the bilayer stabilizingcomponent. It will be apparent to those of skill in the art that incontrast to the phosphatidylethanoiamines, ceramides have only one acylgroup which can be readily varied in terms of its chain length anddegree of saturation. Ceramides suitable for use in accordance with thepresent invention are commercially available. In addition, ceramides canbe isolated, for example, from egg or brain using well-known isolationtechniques or, alternatively, they can be synthesized using the methodsand techniques disclosed in U.S. patent application Ser. No. 08/316,429,filed Sep. 30, 1994, and U.S. Patent Application filed on an even dateherewith and bearing Attorney Docket No. 16303-001010, the teachings ofwhich are incorporated herein by reference. Using the synthetic routesset forth in the foregoing application, ceramides having saturated orunsaturated fatty acids with carbon chain lengths in the range of C₂ toC₃₁ can be prepared.

In addition to the foregoing, detergents, proteins and peptides can beused as bilayer stabilizing components. Detergents which can be used asbilayer stabilizing components include, but are not limited to, TritonX-100, deoxycholate, octylglucoside and lyso-phosphatidylcholine.Proteins which can be used as bilayer stabilizing components include,but are not limited to, glycophorin and cytochrome oxidase. Cleavage ofthe protein, by endogenous proteases, resulting in the loss of thebullcy domain external to the bilayer would be expected to reduce thebilayer stabilizing ability of the protein. In addition, peptides whichcan be used as bilayer stabilizing components include, for example, thepentadecapeptide, alanine-(aminobutyric acid-alanine)₁₄. This peptidecan be coupled, for example, to polyethyleneglycol which would promoteits transfer out of the bilayer. Alternatively, peptides such ascardiotoxin and melittin, both of which are known to induce non-lamehlarphases in bilayers, can be coupled to PEG and might thereby be convertedto bilayer stabilizers in much the same way that PE is converted from anon-lamellar phase preferring lipid to a bilayer stabilizer when it iscoupled to PEG. If the bond between the peptide and the PEG is labile,then cleavage of the bond would result in the loss of the bilayerstabilizing ability and in the restoration of a non-lamellar phase,thereby causing the liposome to become fusogenic.

Typically, the bilayer stabilizing component is present at aconcentration ranging from about 0.05 mole percent to about 50 molepercent. In a presently preferred embodiment, the bilayer stabilizingcomponent is present at a concentration ranging from 0.05 mole percentto about 25 mole percent. In an even more preferred embodiment, thebilayer stabilizing component is present at a concentration ranging from0.05 mole percent to about 15 mole percent. One of ordinary skill in theart will appreciate that the concentration of the bilayer stabilizingcomponent can be varied depending on the bilayer stabilizing componentemployed and the rate at which the liposome is to become fusogenic.

By controlling the composition and concentration of the bilayerstabilizing component, one can control the rate at which the bilayerstabilizing component exchanges out of the liposome and, in turn, therate at which the liposome becomes fusogenic. For instance, when apolyethyleneglycol-phosphatidylethanolamine conjugate or apolyethyleneglycol-ceramide conjugate is used as the bilayer stabilizingcomponent, the rate at which the liposome becomes fusogenic can bevaried, for example, by varying the concentration of the bilayerstabilizing component, by varying the molecular weight of thepolyethyleneglycol, or by varying the chain length and degree ofsaturation of the acyl chain groups on the phosphatidylethanolamine orthe ceramide. In addition, other variables including, for example, pH,temperature, ionic strength, etc. can be used to vary and/or control therate at which the liposome becomes fusogenic. Other methods which can beused to control the rate at which the liposome becomes fusogenic willbecome apparent to those of skill in the art upon reading thisdisclosure.

In a presently preferred embodiment, the fusogenic liposomes containcholesterol. It has been determined that when cholesterol-free liposomesare used in vivo, they have a tendency to absorb cholesterol from plasmalipoproteins and cell membranes. Since this absorption of cholesterolcould, in theory, change the fusogenic behavior of the liposomes,cholesterol can be included in the fusogenic liposomes of the presentinvention so that little or no net transfer of cholesterol occurs invivo. Cholesterol, if included, is generally present at a concentrationranging from 0.02 mole percent to about 50 mole percent and, morepreferably, at a concentration ranging from about 35 mole percent toabout 45 mole percent.

A variety of methods are available for preparing liposomes as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO91\17424, Deamer and Bangham, Biochim. Biophys. Acta, 443:629-634(1976); Fraley, et al., Proc. Natl. Acad. Sci. USA 76:3348-3352 (1979);Hope, et al., Biochim. Biophys. Acta 812:55-65 (1985); Mayer, et al.,Biochim. Biophys. Acta 858:161-168 (1986); Williams, et al., Proc. Natl.Acad. Sci. USA 85:242-246 (1988); the text Liposomes, (Marc J. Ostro(ed.), Marcel Dekker, Inc., New York, 1983, Chapter 1); and Hope, etal., Chem. Phys. Lip. 40:89 (1986), all of which are incorporated hereinby reference. Suitable methods include, for example, sonication,extrusion, high pressure/homogenization, microfluidization, detergentdialysis, calcium-induced fusion of small liposome vesicles andether-fusion methods, all of which are well known in the art. One methodproduces multilamellar vesicles of heterogeneous sizes. In this method,the vesicle-forming lipids are dissolved in a suitable organic solventor solvent system and dried under vacuum or an inert gas to form a thinlipid film. If desired, the film may be redissolved in a suitablesolvent, such as tertiary butanol, and then lyophilized to form a morehomogeneous lipid mixture which is in a more easily hydrated powder-likeform. This film is covered with an aqueous buffered solution and allowedto hydrate, typically over a 15-60 minute period with agitation. Thesize distribution of the resulting multilamellar vesicles can be shiftedtoward smaller sizes by hydrating the lipids under more vigorousagitation conditions or by adding solubilizing detergents such asdeoxycholate.

Unilamellar vesicles are generally prepared by sonication or extrusion.Sonication is generally preformed with a tip sonifier, such as a Bransontip sonifier, in an ice bath. Typically, the suspension is subjected toseveral sonication cycles. Extrusion can be carried out by biomembraneextruders, such as the Lipex Biomembrane Extruder. Defined pore size inthe extrusion filters can generate unilamellar liposomal vesicles ofspecific sizes. The liposomes can also be formed by extrusion through anasymmetric ceramic filter, such as a Ceraflow Microfilter, commerciallyavailable from the Norton Company, Worcester Mass.

Following liposome preparation, the liposomes may be sized to achieve adesired size range and relatively narrow distribution of liposome sizes.A size range of about 0.05 microns to about 0.20 microns allows theliposome suspension to be sterilized by filtration through aconventional filter, typically a 0.22 micron filter. The filtersterilization method can be carried out on a high through-put basis ifthe liposomes have been sized down to about 0.05 microns to about 0.20microns.

Several techniques are available for sizing liposomes to a desired size.One sizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles less than about 0.05 microns in size.Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. In both of these methods, theparticle size distribution can be monitored by conventional laser-beamparticle size discrimination. In addition, the size of the liposomalvesicle can be determined by quasi-electric light scattering (QELS) asdescribed in Bloomfield, Ann. Rev. Biophys. Bioeng. 10:421-450 (1981),incorporated herein by reference. Average liposome diameter can bereduced by sonication of formed liposomes. Intermittent sonicationcycles can be alternated with QELS assessment to guide efficientliposome synthesis.

Extrusion of liposome through a small-pore polycarbonate membrane or anasymmetric ceramic membrane is also an effective method for reducingliposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in liposome size. For use in the presentinventions, liposomes having a size of from about 0.05 microns to about0.45 microns are preferred.

For the delivery of therapeutic agents, the fusogenic liposomes of thepresent invention can be loaded with a therapeutic agent andadministered to the subject requiring treatment. The therapeutic agentswhich can be administered using the fusogenic liposomes of the presentinvention can be any of a variety of drugs, peptides, proteins, DNA, RNAor other bioactive molecules. Moreover, cationic lipids may be used inthe delivery of therapeutic genes or oligonucleotides intended to induceor to block production of some protein within the cell. Nucleic acid isnegatively charged and must be combined with a positively charged entityto form a complex suitable for formulation and cellular delivery.

Cationic lipids have been used in the transfection of cells in vitro andin vivo (Wang, C-Y, Huang L., “pH sensitive immunoliposomes mediatetarget cell-specific delivery and controlled expression of a foreigngene in mouse,” Proc. Natl. Acad. Sci. USA, 1987; 84:7851-7855 and Hyde,S. C., Gill, D. R., Higgins, C. F., et al., “Correction of the iontransport defect in cystic fibrosis transgenic mice by gene therapy,”Nature, 1993; 362:250-255). The efficiency of this transfection hasoften been less than desired, for various reasons. One is the tendencyfor cationic lipids complexed to nucleic acid to form unsatisfactorycarriers. These carriers are improved by the inclusion of PEG lipids.

Cationic lipids useful in producing lipid based carriers for gene andoligonucleotide delivery include, but are not limited to,3β-(N-(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE); diheptadecylamidoglycyl spermidine (DOGS);N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (DOSPA);N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); LIPOFECTIN, acommercially available cationic lipid comprising DOTMA and DOPE(GIBCO/BRL, Grand Island, N.Y.) (U.S. Pat. Nos. 4,897,355; 4,946,787;and 5,208,036 issued to Epstein, et al.); LIPOFECTACE or DDAB(dimethyldioctadecyl ammonium bromide) (U.S. Pat. No. 5,279,883 issuedto Rose); LIPOFECTAMINE, a commercially available cationic lipidcomposed of DOSPA and DOPE (GIBCO/BRL, Grand Island, N.Y.); TRANSFECTAM,a commercially available cationic lipid comprising DOGS (Promega Corp.,Madison, Wis.).

Any variety of drugs which are selected to be an appropriate treatmentfor the disease to be treated in the tissue can be administered usingthe fusogenic liposomes of the present invention. Often the drug will bean antineoplastic agent, such as vincristine, doxorubicin, cisplatin,bleomycin, cyclophosphamide, methotrexate, streptozotocin, and the like.It may also be desirable to deliver anti-infective agents to specifictissues by the present methods. The compositions of the presentinvention can also be used for the selective delivery of other drugsincluding, but not limited to local anesthetics, e.g., dibucaine andchlorpromazine; beta-adrenergic blockers, e.g., propranolol, timolol andlabetolol; antihypertensive agents, e.g., clonidine and hydralazine;anti-depressants, e.g., imipramine, amitriptyline and doxepim;anti-convulsants, e.g., phenytoin; antihistamines, e.g.,diphenhydramine, chlorphenirimine and promethazine; antibacterialagents, e.g., gentamycin; antifungal agents, e.g., miconazole,terconazole, econazole, isoconazole, butaconazole, clotrimazole,itraconazole, nystatin, naftifine and amphotericin B; antiparasiticagents, hormones, hormone antagonists, immunomodulators,neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics,and imaging agents. Other particular drugs which can be selectivelyadministered by the compositions of the present invention will be wellknown to those of skill in the art. Additionally, two or moretherapeutic agents may be administered simultaneously if desired, wheresuch agents produce complementary or synergistic effects.

Methods of loading conventional drugs into liposomes include anencapsulation technique and the transmembrane potential loading method.In one encapsulation technique, the drug and liposome components aredissolved in an organic solvent in which all species are miscible andconcentrated to a dry film. A buffer is then added to the dried film andliposomes are formed having the drug incorporated into the vesiclewalls. Alternatively, the drug can be placed into a buffer and added toa dried film of only lipid components. In this manner, the drug willbecome encapsulated in the aqueous interior of the liposome. The bufferwhich is used in the formation of the liposomes can be any biologicallycompatible buffer solution of, for example, isotonic saline, phosphatebuffered saline, or other low ionic strength buffers. Generally, thedrug will be present in an amount of from about 0.01 ng/mL to about 50mg/mL. The resulting liposomes with the drug incorporated in the aqueousinterior or in the membrane are then optionally sized as describedabove.

Transmembrane potential loading has been described in detail in U.S.Pat. No. 4,885,172, U.S. Pat. No. 5,059,421, and U.S. Pat. No.5,171,578, the contents of which are incorporated herein by reference.Briefly, the transmembrane potential loading method can be used withessentially any conventional drug which exhibits weak acid or weak basecharacteristics. Preferably, the drug will be relatively lipophilic sothat it will partition into the liposome membrane. A pH gradient iscreated across the bilayers of the liposomes or protein-liposomecomplexes, and the drug is loaded into the liposome in response to thepH gradient. The pH gradient is generated by creating a proton gradientacross the membrane either by making the interior more acidic or basicthan the exterior (Harrigan, et al., Biochem. Biophys. Acta.1149:329-339 (1993), the teachings of which are incorporated herein byreference), or by establishing an ion gradient employing ionizableagents, such as ammonium salts, which leads to the generation of a pHgradient (See, U.S. Pat. No. 5,316,771 (Barenholz), the teachings ofwhich are incorporated herein by reference).

In certain embodiments of the present invention, it is desirable totarget the liposomes of the invention using targeting moieties that arespecific to a particular cell type, tissue, and the like. Targeting ofliposomes using a variety of targeting moieties (e.g., ligands,receptors and monoclonal antibodies) has been previously described (see,e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044, both of which areincorporated herein by reference).

Examples of targeting moieties include monoclonal antibodies specific toantigens associated with neoplasms, such as prostate cancer specificantigen. Tumors can also be diagnosed by detecting gene productsresulting from the activation or overexpression of oncogenes, such asras or c-erB2. In addition, many tumors express antigens normallyexpressed by fetal tissue, such as the alphafetoprotein (AFP) andcarcinoembryonic antigen (CEA). Sites of viral infection can bediagnosed using various viral antigens such as hepatitis B core andsurface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virusantigens, human immunodeficiency type-1 virus (HIV1) and papilloma virusantigens. Inflammation can be detected using molecules specificallyrecognized by surface molecules which are expressed at sites ofinflammation such as integrins (e.g., VCAM-1), selectin receptors (e.g.,ELAM-1) and the like.

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes lipidcomponents, e.g., phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid derivatized bleomycin. Antibody targeted liposomes can beconstructed using, for instance, liposomes which incorporate protein A(see, Renneisen, et al., J. Biol. Chem., 265:16337-16342 (1990) andLeonetti, et al., Proc. Natl. Acad. Sci. (USA) 87:2448-2451 (1990), bothof which are incorporated herein by reference).

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The liposome is typically fashioned insuch a way that a connector portion is first incorporated into themembrane at the time of forming the membrane. The connector portion musthave a lipophilic portion which is firmly embedded and anchored in themembrane. It must also have a hydrophilic portion which is chemicallyavailable on the aqueous surface of the liposome. The hydrophilicportion is selected so that it will be chemically suitable to form astable chemical bond with the targeting agent which is added later.Therefore, the connector molecule must have both a lipophilic anchor anda hydrophilic reactive group suitable for reacting with the target agentand holding the target agent in its correct position, extended out fromthe liposome's surface. In some cases it is possible to attach thetarget agent to the connector molecule directly, but in most instancesit is more suitable to use a third molecule to act as a chemical bridge,thus linking the connector molecule which is in the membrane with thetarget agent which is extended, three dimensionally, off of the vesiclesurface.

Following a separation step as may be necessary to remove free drug fromthe medium containing the liposome, the liposome suspension is broughtto a desired concentration in a pharmaceutically acceptable carrier foradministration to the patient or host cells. Many pharmaceuticallyacceptable carriers may be employed in the compositions and methods ofthe present invention. Suitable formulations for use in the presentinvention are found in Remington's Pharmaceutical Sciences, MackPublishing Company, Philadelphia, Pa., 17th ed. (1985). A variety ofaqueous carriers may be used, for example, water, buffered water, 0.4%saline, 0.3% glycine, and the like, and may include glycoproteins forenhanced stability, such as albumin, lipoprotein, globulin, etc.Generally, normal buffered saline (135-150 mM NaCl) will be employed asthe pharmaceutically acceptable carrier, but other suitable carrierswill suffice. These compositions can be sterilized by conventionalliposomal sterilization techniques, such as filtration. The compositionsmay contain pharmaceutically acceptable auxiliary substances as requiredto approximate physiological conditions, such as pH adjusting andbuffering agents, tonicity adjusting agents, wetting agents and thelike, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc. These compositions can be sterilized usingthe techniques referred to above or, alternatively, they can be producedunder sterile conditions. The resulting aqueous solutions may bepackaged for use or filtered under aseptic conditions and lyophilied,the lyophilized preparation being combined with a sterile aqueoussolution prior to administration.

The concentration of liposomes in the carrier may vary. Generally, theconcentration will be about 20-200 mg/ml, usually about 50-150 mg/ml,and most usually about 75-125 mg/ml, e.g., about 100 mg/ml. Persons ofskill may vary these concentrations to optimize treatment with differentliposome components or for particular patients. For example, theconcentration may be increased to lower the fluid load associated withtreatment.

The present invention also provides methods for introducing therapeuticcompounds into cells of a host. The methods generally compriseadministering to the host a fusogenic liposome containing thetherapeutic compound, wherein the fusogenic liposome comprises a bilayerstabilizing component and a lipid which adopts a non-lamellar phaseunder physiological conditions, yet which is capable of assuming abilayer structure in the presence of said bilayer stabilizing component.The host may be a variety of animals, including humans, non-humanprimates, avian species, equine species, bovine species, swine,lagomorpha, rodents, and the like.

The cells of the host are usually exposed to the liposomal preparationsof the invention by in vivo administration of the formulations, but exvivo exposure of the cells to the liposomes is also feasible. In vivoexposure is obtained by administration of the liposomes to host. Theliposomes may be administered in many ways. These include parenteralroutes of administration, such as intravenous, intramuscular,subcutaneous, and intraarterial. Generally, the liposomes will beadministered intravenously or in some cases via inhalation. Often, theliposomes will be administered into a large central vein, such as thesuperior vena cava or inferior vena cava, to allow highly concentratedsolutions to be administered into large volume and flow vessels. Theliposomes may be administered intraarterially following vascularprocedures to deliver a high concentration directly to an affectedvessel. In some instances, the liposomes may be administered orally ortransdermally, although the advantages of the present invention are bestrealized by parenteral administration. The liposomes may also beincorporated into implantable devices for long duration releasefollowing placement.

As described above, the liposomes will generally be administeredintravenously or via inhalation in the methods of the present invention.Often multiple treatments will be given to the patient. The dosageschedule of the treatments will be determined by the disease and thepatient's condition. Standard treatments with therapeutic compounds thatare well known in the art may serve as a guide to treatment withliposomes containing the therapeutic compounds. The duration andschedule of treatments may be varied by methods well known to those ofskill, but the increased circulation time and decreased in liposomeleakage will generally allow the dosages to be adjusted downward fromthose previously employed. The dose of liposomes of the presentinvention may vary depending on the clinical condition and size of theanimal or patient receiving treatment. The standard dose of thetherapeutic compound when not encapsulated may serve as a guide to thedose of the liposome-encapsulated compound. The dose will typically beconstant over the course of treatment, although in some cases the dosemay vary. Standard physiological parameters may be assessed duringtreatment that may be used to alter the dose of the liposomes of theinvention.

Liposome charge is an important determinant in liposome clearance fromthe blood, with negatively charged liposomes being taken up more rapidlyby the reticuloendothelial system (Juliano, Biochem. Biophys. Res.Commun. 63:651 (1975)) and, thus, having shorter half-lives in thebloodstream. Liposomes with prolonged circulation half-lives aretypically desirable for therapeutic and diagnostic uses. To maximizecirculation half-lives, the bilayer stabilizing component should be ahydrophilic polymer, e.g., PEG, conjugated to lipid anchors, e.g., PEs,having long, saturated hydrocarbon chains (C18>C16>C14) as theseconjugates provide a longer lasting steric barrier. As such, by varyingthe charge in addition to the foregoing factors, one of skill in the artcan regulate the rate at which the liposomes of the present inventionbecome fusogenic.

Additionally, the liposome suspension may include lipid-protectiveagents which protect lipids against free-radical and lipid-peroxidativedamages on storage. Lipophilic free-radical quenchers, such asalphatocopherol and water-soluble iron-specific chelators, such asferrioxamine, are suitable.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner.

EXAMPLES I. Materials and General Methods

A. Materials

All phospholipids including fluorescent probes and PEG-PE conjugateswere purchased from Avanti Polar Lipids, Birmingham, Ala., USA.1-O-methyl-(poly(ethoxy)-O-succinyl-O-(egg)ceramide which was a giftfrom Dr L. Choi of Inex Pharmaceuticals Corp., Vancouver, BC, Canada.Di-[1-¹⁴C]-palmitoylphosphatidyl-choline was purchased from Du Pont,Mississuaga, Ont., Canada. [³H]-DSPE-PEG₂₀₀₀ was synthesized asdescribed previously (Parr, et al., Biochim. Biophys. Acta, 1195: 21-30(1994)). Other reagents were purchased from Sigma Chemical Co., StLouis, Mo., USA.

B. Preparation of Multilamellar Vesicles and Large Unilamellar Vesicles

Lipid components were mixed in 1-2 ml of benzene:methanol (95:5, v/v)and then lyophilized for a minimum of 5 hours at a pressure of <60millitorr using a Virtis lyophilizer equipped with a liquid N₂ trap.Multilamellar vesicles (MLVs) were prepared by hydrating the dry lipidmixtures in 150 mM NaCl, buffered with 10 mM Hepes-NaOH, pH 7.4(Hepes-buffered saline, HBS). Mixtures were vortexed to assisthydration. To produce large unilamellar vesicles (LUVs), MLVs were firstfrozen in liquid nitrogen and then thawed at 30° C. five times. LUVswere produced by extrusion of the frozen and thawed MLVs ten timesthrough 2 stacked polycarbonate filters of 100 nm pore size at 30° C.and pressures of 200-500 psi (Hope, et al., Biochim. Biophys. Acta,812:55-65 (1985)).

C. ³¹P-NMR Spectroscopy

³¹P-NMR spectra were obtained using a temperature controlled BrukerMSL200 spectrometer operating at 81 MHz. Free induction decays wereaccumulated for 2000 transients using a 4 μs, 90° pulse, 1 sec.interpulse delay, 20 KHz sweep width and Waltz decoupling. A 50 Hz linebroadening was applied to the data prior to Fourier transformation.Samples were allowed to equilibrate at the indicated temperature for 30minutes prior to data accumulation. Lipid concentrations of 30-70 mMwere used.

D. Freeze-fracture Electron Microscopy

MLVs were prepared by hydrating a mixture ofDOPE:cholesterol:DOPE-PEG₂₀₀₀ (1:1:0:1) with HBS. A portion of themixture was extruded as described above to produce LUVs. Glycerol wasadded to both MLVs and LUVs to a final concentration of 25% and sampleswere rapidly frozen in liquid freon. The samples were fractured at −110°C. and <10⁻⁶ torr in a Balzers BAF400 unit. Replicas were prepared byshadowing at 45° with a 2 nm layer of platinum and coating at 90° with a20 nm layer of carbon. The replicas were cleaned by soaking inhypochlorite solution for up to 48 hrs and were visualized in a JeolJEM-1200 EX electron microscope.

E. Gel Filtration of LUVs and Micelles

LUVs composed of DOPE:cholesterol:DSPE-PEG₂₀₀₀ (1:1:0:1) with traceamounts of ¹⁴C-DPPC and ³H-DSPE-PEG₂₀₀₀ were chromatographed at a flowrate of approximately 0.5 ml/min on a column of Sepharose CL-4B waspretreated with 10 mg of eggPC, which had been suspended in HBS by bathsonication, to eliminate non-specific adsorption of lipid to the column.Micelles were prepared by hydrating DSPE-PEG₂₀₀₀ containing a traceamount of ³H-DSPE-PEG₂₀₀₀ with HBS and chromatographed as described forLUVs.

F. Lipid Mixing Assays

Lipid mixtures were prepared as described for NMR measurements. Theresultant multilamellar vesicles (MLV) were frozen in liquid nitrogenand then thawed at 30° C. five times. Large unilamellar vesicles (LUV)were produced by extrusion of the frozen and thawed MLV ten timesthrough 2 stacked polycarbonate filters of 100 mn pore size at 30° C.and pressures of 200-500 psi (Hope, et al., Biochim. Biophys. Acta812:55-65 (1985)).

Lipid mixing was measured by a modification of the fluorescenceresonance energy transfer (FRET) assay of Struck, et al. (Biochemistry20:4093-4099 (1981)). LUVs were prepared containing the fluorescentlipids,N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dioleoylphosphatidylethanolamine(NBD-PE) and N-(lissamine rhodamine Bsulfonyl)-dipalmitoylphosphatidylethanolamine (Rh-PE) at 0.5 mol %. LUVs(50-60 μM) and a three-fold excess of unlabelled target vesicles weremixed in the fluorimeter at 37° C. for short term assays (≦1 hour), orin sealed cuvettes in a dark water bath at 37° C. for longer assays. Formeasurements of fusion after PEG-lipid transfer, an excess of liposomesprepared from 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) was addedas a sink for the PEG-lipid. Fluorescence emission intensity wasmeasured at 517 nm with excitation at 465 nm both before and after theaddition of Triton X-100 (final concentration of 0. 5% or 1% when POPCsink was used). Data is presented as either uncorrected fluorescenceintensity for short term assays (<1 hour) or as percentage fusion. Lightscattering controls were performed by replacing LUVs labelled with 0.5mol % probes with unlabelled vesicles. Maximum fusion was determinedusing mock fused vesicles containing 0.125 mol % of each fluorescentprobe. The percentage fusion was calculated as:${\% \quad {Fusion}} = {\frac{\frac{( {F_{(t)} - L_{(t)}} )}{( {F_{T} - L_{T}} )} - \frac{( {F_{O} - L_{O}} )}{( {F_{T} - L_{T}} )}}{\frac{( {M_{(t)} - L_{(t)}} )}{( {M_{T} - L_{T}} )} - \frac{( {F_{O} - L_{O}} )}{( {F_{T} - L_{T}} )}} \times 100}$

where F_((t))=fluorescence intensity at time t; F_(o)=fluorescenceintensity at zero time; F_(T)=fluorescence intensity in the presence ofTriton X-100. M and L represent the same measurements for the mock fusedcontrol and the light scattering control respectively. Changes influorescence of the mock fused control indicated that exchange of thefluorescent probes over 24 hours accounted for 10% of the fluorescencechange observed, but was negligible over the first hour.

G. Fusion of Liposomes with Red Blood Cells

LUVs composed of DOPE:cholesterol:DODAC (40:45:15) orDOPE:cholesterol:DODAC:PEG-ceramide (35:45:15:) were prepared bystandard extrusion techniques. LUVs also contained 1 mol % rhodamine-PE.LUVs (200 /M) were incubated at 37° C. with 50 μl packed RBCs in a finalvolume of 1 ml. For assays of fusion after PEG-lipid exchange, a sink of2 mM POPC:cholesterol (55:45) was included. In some assays, thefusogenic liposomes were pre-incubated with the sink before being mixedwith the RBCs (See, figure legends for FIGS. 22-24). Aliquots of themixtures were transferred to glass microscope slides, covered with coverslips and examined by phase contrast and fluorescent microscopy. Fusionwas assessed as fluorescent labeling of the RBC plasma membranes. ForFIGS. 22-24, fluorescent liposomes were incubated with POPC:cholesterolliposomes and/or RBCs as described in section “L,” infra. Panels a,c ande of FIGS. 22-24 are views under phase contrast, whereas panels b,d andf of FIGS. 22-24 are the same fields viewed under fluorescent light.

H. Other Procedures

Phospholipid concentrations were determined by assaying for phosphateusing the method of Fiske and Subbarow (J. Biol. Chem., 66:375-400(1925)). Liposome size distributions were determined by quasi-elasticlight scattering (QELS) using a Nicomp model 370 particle sizer.

II. Experimental Findings

A. Influence of BSC on the Polymorphic Phase Properties of an EquimolarMixture of DOPE and Cholesterol

³¹P-NMR was used to examine the effect of bilayer stabilizing component(BSC), in this instance poly-(ethyleneglycol)₂₀₀₀ conjugated to DOPE(i.e., DOPE-PEG₂₀₀₀), on the phase preference of an equimolar mixture ofDOPE and cholesterol (FIG. 1). In the absence of BSC, the mixtureadopted an inverse hexagonal phase (H_(II)) at 20° C. as determined fromthe characteristic ³¹P-NMR lineshape with a low field peak and highfield shoulder (Cullis and deKruijff, Biochim. Blophys. Acta 559:399-420(1979)). As the amount of BSC in the mixture was increased, the peakcorresponding to H_(II) phase phospholipid disappeared and a high fieldpeak with a low field shoulder, characteristic of bilayer phasephospholipid (Cullis and deKruijff, supra, 1979) appeared. WhenDOPE-PEG₂₀₀₀ was present at 20 mol % of phospholipid, the mixture wasalmost exclusively bilayer with no evidence of H_(II), phase lipid.

In addition to the peaks corresponding to H_(II) phase and bilayerphase, a third peak indicative of isotropic motional averaging wasobserved in the presence of BSC (FIG. 1). The size of the isotropicsignal varied with the amount of BSC present and, as shown in subsequentFigures, the nature of the BSC species. The signal was largest atconcentrations of BSC that allowed H_(II) and bilayer phases to co-existand diminished when either H_(II) or bilayer phase predominated. Such asignal may be produced by a number of phospholipid phases which allowisotropic motional averaging on the NMR timescale, including micellar,small vesicular, cubic and rhombic phase phospholipids.

B. The Influence of BSC on the Thermotropic Properties of an EquimolarMixture of DOPE and Cholesterol

FIG. 2 illustrates the effect of temperature on the phase properties ofmixtures of DOPE, cholesterol and BSC. When DOPE-PEG₂₀₀₀ was present at9 mol %, there was a large isotropic signal which dominated the spectrumat all temperatures. The predominant, non-isotropic phase at 0° C. wasbilayer. However, as the temperature was increased the high field peakdiminished and a shoulder corresponding to the low field peak of theH_(II) phase appeared. The apparent bilayer to hexagonal phasetransition occurred at 40-50° C., but was almost obscured by the largeisotropic signal. DOPE on its own exhibits a sharp transition over aninterval of approximately 10° C. (see, FIG. 1 in Tilcock, et al.,Biochemistry 21:4596-4601 (1982)). The transition in mixtures of DOPE,cholesterol and BSC was slow in comparison with both phases present overa temperature range of almost 40° C. (See also, FIG. 3).

The mixture was stabilized in the bilayer conformation over the sametemperature range when the BSC content was increased to 20 mol % (FIG.2). There was no evidence of phospholipid in the H_(II) phase. Inaddition, the isotropic signal was markedly reduced at the higher BSCconcentration at all temperatures studied. The amount of lipidexperiencing isotropic motional averaging increased as the temperatureincreased for both concentrations of BSC.

C. The Effect of Head Group Size on the Bilayer Stabilizing Propertiesof BSCs

The influence of head group size on the bilayer stabilizing propertiesof BSCs is illustrated in FIG. 3. DOPE-PEG₂₀₀₀ at 5 mol % had limitedbilayer stabilizing ability. A broad bilayer to H_(II) transition wascentered at approximately 10° C., but a large proportion of the lipidadopted non-bilayer phases at all temperatures examined. Increasing thesize of the headgroup by using poly-(ethyleneglycol)₅₀₀₀ conjugated toDOPE (DOPE-PEG₅₀₀₀) in place of DOPE-PEG₂₀₀₀, at the same molarfraction, caused a marked increase in bilayer stability. The bilayer toH_(II) transition temperature increased to approximately 30° C. and theisotropic signal was barely discernible. The broadening of the bilayerto H_(II) transition noted above is particularly evident here withH_(II) phase lipid present at 0° C. and bilayer phase lipid present at40° C.

D. The Influence of Acyl Chain Composition on the Bilayer StabilizingProperties of BSCs

The bilayer stabilizing ability of three BSCs differing only in acylchain composition is shown in FIG. 4. PEG₂₀₀₀ conjugated todimyristoylphosphatidyl-ethanolamine (DMPE-PEG₂₀₀₀),dipalmitoylphosphatidylethanolamine (DPPE-PEGM₂₀₀₀) ordistearoylphosphatidylethanolamine (DSPE-PEG₂₀₀₀) showed a similarability to stabilize an equimolar mixture of DOPE and cholesterol. Thebilayer to H_(II) phase transition was raised to approximately 40-50° C.The results are similar to those presented in FIG. 2 which were obtainedusing a BSC with the same headgroup, but unsaturated acyl groups(DOPE-PEG₂₀₀₀ at the same concentration. The size of the isotropicsignal varied somewhat with the different BSCs, being smallest withDSPE-PEG₂₀₀₀ and largest with DOPE-PEG₂₀₀₀ (cf., FIG. 2 and FIG. 4).

E. The Use of PEG-ceramides as Bilayer Stabilizing Components

The spectra set forth in FIGS. 1-4 were all obtained using PEGconjugated to phosphatidylethanolamine through a carbamate linkage. Inaddition, however, the use of ceramide as an alternative anchor for thehydrophilic polymer was examined. PEG₂₀₀₀ was conjugated via a succinatelinker to egg ceramide. FIG. 5 shows the ³¹P-NMR spectra obtained usingmixtures of DOPE:cholesterol:egg ceramide-PEG₂₀₀₀ (1:1:0.1 and 1:1:0.25)over the temperature range of 0 to 60° C. At the lower molar ratio ofPEG-ceramide, both bilayer and H_(II) phase lipid are in evidence atmost temperatures. However, at the higher PEG-ceramide molar ratio, thespectra are exclusively bilayer up to 60° C. at which point a low fieldshoulder corresponding to H_(II) phase lipid is visible. Unlike thespectra obtained using PEG-PEs, there was almost no isotropic signalwhen PEG-ceramide was used.

F. Freeze-fracture Electron Microscopy

One of the interesting features of several of the NMR spectra was thenarrow signal at 0 ppm, indicative of isotropic motional averaging. Thissignal can arise from a number of phospholipid phases such as micellar,small vesicular, cubic and rhombic phase structures. Freeze-fractureelectron microscopy was used to investigate this aspect further. FIG. 6shows an electron micrograph of MLVs prepared by hydrating a mixture ofDOPE:cholesterol:DOPE-PEG₂₀₀₀ (1:1:0.1) with HBS at room temperature.This lipid mixture corresponds to the NMR spectra set forth in FIG. 2Awhich exhibited evidence of bilayer, H_(II) and isotropic phases.

A number of different structures are visible in the micrograph. Much ofthe lipid is present as large spherical vesicles of 400 to 600 nm indiameter. Many of the vesicles have indentations which appear to berandomly distributed in some vesicles, but organized in straight orcurved lines in others. Cusp-like protrusions are also visible on theconcave surfaces of some vesicles. These features are commonly referredto as lipidic particles (Verkleij, A. J., Biochim. Biophys. Acta,779:43-92 (1984)) and may represent an intermediate structure formedduring fusion of bilayers. These large vesicles would be expected togive rise to a predominately bilayer ³¹P-NMR spectrum with a narrowisotropic signal due to the lipidic particles. Similar results have beenobserved with N-methylated PEs (Gagne, et al., Biochemistry,24:4400-4408 (1985)). A number of smaller vesicles of around 100 nmdiameter can also be seen. These vesicles may have been formedspontaneously on hydration, or may have been produced by vesiculizationof larger vesicles. These vesicles are sufficiently small for lipidlateral diffusion, or tumbling of the vesicles in suspension, to producemotional averaging on the NMR timescale (Bumell, et al., Biochim.Biophys. Acta, 603:63-69 (1980)), giving rise to an isotropic signal(see, FIG. 2A). In the center of FIG. 6 is a large aggregate showingevidence of several different structures. The right side of theaggregate is characterized by what appears to be closely packed lipidicparticles. The upper left hand side shows a distinct organization intothree-dimensional cubic arrays and the lower left hand region has theappearance of stacked tubes characteristic of lipid adopting the Haphase (Hope, et al., J. Elect. Micros. Tech., 13:277-287 (1989)). Thisis consistent with the corresponding ³¹P-NMR spectrum.

FIG. 7 shows the appearance of the same mixture after extrusion throughpolycarbonate filters of 100 nm pore size to produce LUVS. The lipid ispredominately organized into vesicles of approximately 100 nm indiameter. Closer inspection reveals the presence of occasional largervesicles and some of tubular shape. Overall the fairly uniform sizedistribution is typical of that obtained when liposomes are produced byextrusion.

The presence of lipid micelles is not readily apparent from freezefracture electron microscopy. Lipid in the micellar phase could,however, contribute to the isotropic signal observed in NMR spectra, andit has previously been shown that PEG-PE conjugates form micelles whenhydrated in isolation (Woodle and Lasic, Biochim. Biophys. Acta,113:171-199 (1992)). As such, the presence of micelles was tested bysubjecting a suspension of LUVs to molecular sieve chromatography onSepharose 4B. The liposomes were of the same composition used for thefreeze fracture studies above except that DSPE-PEG₂₀₀₀ was used in placeof DOPE-PEG₂₀₀₀, and they contained trace amounts of ¹⁴C-DPPC and³H-DSPE-PEG₂₀₀₀. The elution profile is shown in FIG. 8. A single peakcontaining both the phospholipid and PEG-PE conjugate markers was foundin the void volume. A control experiment also shown in FIG. 8demonstrated that micelles, which formed when PEG-PE was hydrated inisolation, were included into the column and would have been clearlyresolved if present in the liposomal preparation.

G. Effect of PE-PEG₂₀₀₀ On Fusion Of PE:PS LUVs

When unlabelled LUVs composed of DOPE:POPS (1:1) were added tofluorescently labelled LUVs there was a small jump in fluorescenceintensity due to increased light scattering but no fusion (FIG. 9, tracea). Upon addition of 5 mM Ca²⁺, there was a rapid increase influorescence consistent with lipid mixing as a result of membranefusion. Fusion was complete within a few seconds and was followed by aslow decrease in fluorescence. Inspection of the cuvette revealed thepresence of visible aggregates that settled despite stirring, resultingin the observed decrease in fluorescence. When PEG₂₀₀₀ conjugated todimyristoylphosphatidylethanolamine (DMPE-PEG₂₀₀₀) was included in bothvesicle populations, however, inhibition of fusion was observed. Asshown in FIG. 9 (traces b-d), inhibition was dependent on theconcentration of DMPE-PEG₂₀₀₀ in the vesicles with as little as 2 mol %being sufficient to eliminate Ca²⁺-induced fusion.

H. The Effect of PE-PEG Loss on Fusion

Recently, it has been demonstrated that phospholipids conjugated to PEGof molecular weights 750-5,000 Da have enhanced rates of spontaneoustransfer between liposomes. The half-time for transfer of theseconjugates can vary from minutes to hours and depends on both the sizeof the PEG group and the nature of the acyl chains which anchor theconjugate in the bilayer. As such, fusion was examined under conditionswhere the PEG-lipid would be expected to transfer out of the liposomes.Ca²⁺ ions were added to PE:PS liposomes containing 2 mol % DMPE-PEG₂₀₀₀, followed by a twelve-fold excess (over labelled vesicles) of1-paimitoyl-2-oleoyl-phosphatidylcholine (POPC) liposomes as a sink forthe PEG-PE. As shown in FIG. 10 (curve a), while fusion was initiallyblocked by the presence of DMPE-PEG₂₀₀₀, the addition of POPC liposomes,which acted as a sink, lead to recovery of full fusogenic activityfollowing a short time lag. The small initial jump in fluorescenceintensity that occurred when POPC liposomes were added to PE:PSliposomes resulted from increased light scattering, not fusion. Controlexperiments demonstrated that no fusion occurred between the PE:PSliposomes and the POPC liposomes (data not shown), and no fusionoccurred in the absence of POPC liposomes (FIG. 10, curve b).

To confirm that recovery of fusogenic activity was dependent on removalof the PEG-PE, the influence of initial PEG-lipid concentration on theduration of the lag phase prior to fusion was examined (FIG. 11).Liposomes containing equimolar PE and PS were prepared with 2, 3, 5 or10 mol % DMPE-PEG₂₀₀₀. Fluorescently labelled and unlabelled vesicleswere mixed at a ratio of 1:3 and after the addition of 5 mM CaCl₂, a36fold excess (over labelled vesicles) of POPC liposomes was added. Asexpected, there was an increase in the time delay prior to fusion withincreasing PEG-lipid concentration.

I. The Effect of Conjugate Acyl Chain Composition on Fusogenic Activity

Since fusion is dependent on prior transfer of the PEG-PE out of theliposomes, it was thought that the rate at which fusogenic activity wasrecovered would depend on the rate of transfer of the PEG-PE. One factorthat affects the rate at which a phospholipid transfers from onemembrane to another is the length of its acyl chains. As such, theeffect of conjugate acyl chain composition on fusogenic activity wasinvestigated. In doing so, the recovery of fusogenic activity of PE:PSLUVs containing 2 mol % DMPE-PEG₂₀₀₀ was compared with PE:PS LUVscontaining 2 mol % DPPE-PEG₂₀₀₀ and 2 mol % DSPE-PEG₂₀₀₀ (FIG. 12A).Increasing the length of the acyl chains from 14 to 16 carbons caused adramatic increase in the lag period before fusion was initiated.Although the same level of fusion occurred using either DMPE-PEG₂₀₀₀ orDPPE-PEG₂₀₀₀, it was essentially complete in 40 minutes whenDMPE-PEG₂₀₀₀ was the stabilizer, but took 24 hours when DPPE-PEG₂₀₀₀ wasused. The implied decrease in rate of transfer (30-40 fold) isconsistent with previous measurements of the effect of acyl chain lengthon rates of spontaneous phospholipid transfer. Increasing the acyl chainlength to 18 carbons (DSPE-PEG₂₀₀₀, FIG. 12A) extended the lag in fusioneven further and, after 24 hours, the level was only 20% of maximum.

A second factor that affects the rate of spontaneous transfer ofphospholipids between bilayers is the degree of saturation orunsaturation of the acyl chains. The rate of fusion of LUVs containing 2mol % DOPE-PEG₂₀₀₀ is shown in FIG. 12B. The presence of a double bondincreased the rate of recovery of fusogenic activity in the presence ofa sink for the DOPE-PEG₂₀₀₀ over that of the corresponding saturatedspecies (DSPE-PEG₂₀₀₀, FIG. 12A). The rate of fusion was similar to thatseen with DPPE-P₂₀₀₀. FIG. 12B also shows the rate of fusion obtainedwhen the neutral PEG-lipid species, egg ceramide-PEG₂₀₀₀ was used. Therate was somewhat faster than observed with DPPE-PEG₂₀₀₀. Althoughdifferences in the interaction of the two lipid anchors with neighboringphospholipids in the bilayer make direct comparison of interbilayertransfer rates and, hence, fusion difficult, it appears that thepresence of a negative charge on the conjugate (PE-PEG) is not requiredfor desorption of the conjugate from negatively charged bilayers.

J. Effect of PEG Molecular Weight on Fusogenic Activity

The presence of PEG conjugated to the liposome surface results in asteric barrier that inhibits close bilayer apposition and subsequentfusion. The magnitude of the barrier should increase with increasing PEGmolecular weight. When DMPE-PEG₅₀₀₀ was incorporated into PE:PS (1:1)LUVS, a concentration dependent inhibition of fusion was observed (FIG.13A). The results are similar to those obtained with DMPE-PEG₂₀₀₀ (FIG.9), except that only 1 mol % DMPE-PEG₅₀₀₀ was required to completelyinhibit fusion compared to 2 mol % DMPE-PEG₂₀₀₀.

FIG. 13B shows the effect of varying acyl chain composition of thelarger PEG-lipid conjugate on fusion. Interestingly, the rates of fusionobserved with 1 mol % PE-PEG₅₀₀₀ were similar to those with 2 mol %PE-PEG₂₀₀₀. The concentrations used were those shown to be sufficient tocompletely inhibit fusion (cf, FIG. 9 and FIG. 13A). It was thought thatthe larger PEG group would increase the rate of interbilayer transfer ofthe conjugate and, hence, the rate of fusion. However, this was not thecase. To examine this aspect further, the rates of fusion underconditions where the initial surface density of ethylene glycol groupswas similar were compared. FIG. 14 shows the fusion of PE:PS (1:1) LUVscontaining 5 mol % DMPE-PEG₂₀₀₀ or 2 mol % DMPE-PEG₅₀₀₀ after additionof a sink for the PEG-lipid. The rates observed were very similarsuggesting that factors other than loss of the steric barrier as adirect result of interbilayer transfer of the conjugate were involved.

K. Programmable Fusogenic Liposomes Comprising DOPE:cholesterol:DODAC:ceramides

Fluorescently labelled liposomes were prepared in distilled water from amixture of DOPE and N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC)at a molar ratio of 85:15. A three-fold excess of acceptor liposomes ofthe same composition, but containing no fluorescent probes, was added tolabelled liposomes and fusion was initiated after 60s by the addition ofNaCl (FIG. 15). Fusion was highly dependent on ionic strength. Littlefusion was observed at 50 mM NaCl, but with increasing saltconcentration, the rate and extent of fusion increased dramatically. At300 mM NaCl fusion was so extensive that visible aggregates occurred andthese aggregates could not be maintained in suspension resulting in theapparent decrease in fluorescence seen in FIG. 15 for the 300 mM NaClcurve. Importantly, substantial fusion was observed at physiologicalsalt concentration (150 mM).

As described above, the inclusion of 2 mol % PEG-lipid in PE:PSliposomes is sufficient to inhibit Ca²⁺-induced fusion. When 2 mol %DMPE-PEG₂₀₀₀ was included in DOPE:DODAC liposomes(DOPE:DODAC:DMPE-PEG₂₀₀₀, 83:15:2), the same inhibitory effect wasobserved (FIG. 16). However, unlike the PE:PS system, when theseliposomes were incubated for 1 hr in the presence of a large excess ofPOPC liposomes, which acted as a sink for the PEG-PE, little, if any,fusion was observed. Since PEG-PEs are negatively charged thecomplementary charge, interaction with DODAC likely results in adramatic decrease in the rate of transfer out of the bilayer.

As an alternative bilayer stabilizing component, therefore, the abilityof a neutral PEG-lipid species, i.e., PEG-ceramide, to inhibit fusion inthis system was examined. PEG-ceramides have similar bilayer stabilizingproperties to PEG-PEs. For these studies, PEG₂₀₀₀ was conjugated toceramides of various fatty amide chain lengths through a succinatelinker. Liposomes prepared from DOPE:DODAC:(C8:0) ceramide-PEG₂₀₀₀(83:15:2) did not fuse in the presence of 300 mM NaCl. However, when anexcess of POPC liposomes was added, fusion occurred fairly rapidly (FIG.17). Similar results were observed when cholesterol was incorporatedinto the liposomes (DOPE:cholesterol:DODAC:(C8:0) ceramide-PEG₂₀₀₀,38:45:15:2), although the rate of fusion was slower than withcholesterol-free liposomes (FIG. 17).

To determine if the rate of fusion in this system can be controlled, thechain lengths of the fatty amide groups of the PEG-ceramides werevaried. Using a (C14:0) ceramide-PEG₂₀₀₀, 50% maximal fusion wasobserved after approximately 6 hr (FIG. 18). This was a dramaticincrease over the rate with (C8:0) ceramide-PEG₂₀₀₀ shown in FIG. 18,where maximal fusion was achieved in about 40 minutes. The time for 50%maximal fusion was increased to over 20 hr when egg ceramide-PEG₂₀₀₀ wasused. Ceramides derived from egg have a fatty amide chain length ofpredominantly 16:0 (approximately 78%), with small amounts of longersaturated chains. FIG. 18 also shows an extended time course withDMPE-PEG₂₀₀₀. The limited extent of fusion (<20% of maximum at 21 hr)shows the dramatic effect that charge interaction can have on PEG-lipidtransfer rates.

The rationale for using cationic liposomes is that complementary chargeinteraction with anionic plasma membranes will promote association andfusion of liposomes with cells in vivo. It is important, therefore, toconfirm that not only will DOPE:DODAC liposomes fuse with membranescarrying a negative charge, but that incorporation of PEG-lipidconjugates prevents fusion in a programmable manner. This ability isdemonstrated in FIG. 19 which shows that liposomes composed ofDOPE:cholesterol:DODAC, 40:45:15, fuse with negatively charged liposomesand inclusion of a PEG-lipid conjugate in the cationic liposomesinhibits fusion. Fusion between DOPE:DODAC liposomes could be preventedwhen 2 mol % PEG-lipid was present in both fluorescently labelled andacceptor liposomes. When PEG-lipid was omitted from the acceptorliposomes, however, its concentration in the labelled vesicles had to beincreased to 4-5 mol % to block fusion between cationic and anionicliposomes.

Again, while PEG-lipids can inhibit fusion in this system, underconditions where the PEG-lipid can transfer out of the liposomes,fusogenic activity can be restored. FIG. 20 shows that this is, indeed,the case. Incubation of DOPE:cholesterol: DODAC:(C14:0) ceramide-PEG₂₀₀₀(36:45:15:4) liposomes with PE:PS liposomes, in the presence of excessPOPC:cholesterol (55:45) vesicles which act as a sink, results inrecovery of fusogenic activity. In the absence of a sink, a slow rate offusion is observed, indicating that a higher concentration of PEG-lipidis required to completely prevent fusion over longer periods.

While fusion between cationic and anionic liposomes provides a goodmodel system, fusion in vivo is somewhat different. The acceptormembrane is not composed solely of lipid, but contains a highconcentration of proteins, many of which extend outward from the lipidbilayer and may interfere with fusion. Using erythrocyte ghosts as arepresentative membrane system, it was found that liposomes composed ofDOPE:cholesterol:DODAC (40:45:15) fuse with cellular membranes (see,FIG. 21). In addition, it was found that fusion in this system, likethose presented above, can also be inhibited using PEG-lipid conjugates.This results clearly establish the usefulness of these systems asprogrammable fusogenic carriers for intracellular drug delivery.

L. Programmed Fusion with Erythrocytes (RBCs)

LUVs composed of DOPE:cholesterol:DODAC (40:45:15) fused rapidly andextensively with RBCs (FIG. 22, panels a and b). Prolonged incubationcaused extensive lysis of the RBCs and numerous fluorescently labeled“ghosts” were formed. Incorporation of PEG-ceramide (C8:0) at 5 mol %blocked fusion (FIG. 22, panels c and d) and this effect was maintainedfor up to 24 hr. This effect was somewhat surprising since the (C8:0)ceramide can exchange rapidly (i.e., within minutes) between liposomalmembranes. It appears that either the RBCs cannot act as a sink for thePEG-ceramide, or there were insufficient RBCs to remove enoughPEG-ceramide to allow fusion. However, when an exogenous sink for thePEG-ceramide was included, fusogenic activity was recovered withinminutes (FIG. 22, panels e and f).

When PEG-ceramides with longer fatty amide chains (i.e., C14:0 or C20:0)were used, there was little fusion over 24 hr, even in the presence ofan exogenous sink. This again was surprising as substantial fusion isobserved over this time frame in liposomal systems when a sink ispresent. It was thought that some non-specific interaction between thesink (POPC/cholesterol) and the RBCs was occurring which hindered theability of the POPC:cholesterol liposomes to absorb the PEG-ceramide. Toovercome this, the fusogenic liposomes were pre-incubated with the sinkbefore adding RBCS. FIG. 23 shows the results obtained under theseconditions using PEG-ceramide (C14:0). No fusion was observed afterpre-incubation of the fusogenic LUVs with the sink for 5 minutes priorto addition of RBCs (FIG. 23, panels a and b). However, after a 1 hrpre-incubation, some fusion with RBCs was observed (FIG. 23, panels cand d), suggesting that under these conditions the PEG-ceramide couldtransfer out of the liposomes and became fusogenic. With longerincubations (2 hr), the pattern of fluorescent labeling changed. Ratherthan diffuse labeling of the RBC plasma membranes, extensive punctatefluorescence was observed (FIG. 23, panels e and f) and this pattern wasmaintained for up to 24 hr. The punctate fluorescence did not appear tobe associated with cells and it may represent fusion of fluorescentliposomes with the sink, although previous fluorescent measurements ofliposome-liposome fusion indicated that this did not occur to anyappreciable extent. A second possibility is that exchange of thefluorescent probe over the longer time courses leads to labeling of thesink, although it seems unlikely that this would prevent fusion andlabeling of the RBCS. When PEG-ceramide (C20:0) was used, there was noevidence for fusion after preincubation of LUVs with the sink for 5 min(FIG. 24, panels a and b), 1 hr (FIG. 24, panels c and d), 2 hr (FIG.24, panels e and f), or for up to 24 hr (results not shown).

FIGS. 22-24 unequivocally establish that the liposomes of the presentinvention exhibit programmable fusion with intact cells. Firstly,liposomes composed of DOPE:cholesterol:DODAC (40:45:15) that contain noPEG-lipid fuse rapidly and extensively with RBCs. Secondly, when theliposomes contain 5 mol % PEG-lipid fusion is blocked regardless of thecomposition of the lipid anchor. Thirdly, in the presence of a sink towhich the PEG-lipid can transfer, fusogenic activity can be restored ata rate that is dependent on the nature of the lipid anchor. Althoughexchange leading to fusion could not be demonstrated when thePEG-ceramide (C20:0) was used, it is believed this is a problem with theassay rather than a lack of fusogenic potential. In vivo there would bean almost infinite sink for PEG-lipid exchange.

The foregoing is offered for purposes of illustration. It will bereadily apparent to those skilled in the art that the operatingconditions, materials, procedural steps and other parameters of themethods and test devices described herein may be further modified orsubstituted in ways without departing from the spirit and scope of theinvention.

What is claimed is:
 1. A method for increasing the rate of exchange of abilayer stabilizing component from a liposome formulation to improvemultiple dosing, said method comprising: (a) producing a first liposomecomposition having a lipid, a bioactive agent and a first bilayerstabilizing component, wherein said first bilayer stabilizing componentis DSPE-PEG₂₀₀₀; (b) determining the rate of exchange of DSPE-PEG₂₀₀₀from said first liposome composition using a fluorescent lipid mixingassay; and (c) preparing a second liposome formulation nearly identicalto said first liposome formulation having said lipid, said bioactiveagent, but substituting DSPE-PEG₂₀₀₀ from said first liposomecomposition with a second bilayer stabilizing component having a fasterrate of exchanges wherein said second liposome composition results inimproved dosing.
 2. The method of claim 1, wherein said first and secondliposome compositions further comprise cholesterol.
 3. The method ofclaim 1, wherein said lipid is a cationic lipid.
 4. The method of claim3, wherein said cationic lipid is a member selected from the groupconsisting of 3β-(N-(N′,N′-dimethylaminoethane)carbamoyl)cholesterol,N,N-distearyl-N,N-dimethylammonium bromide,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide, N,N-dioleyl-N,N-dimethylammonium chloride,diheptadecylamidoglycyl spermidine,N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniumchloride, N-(1-(2,3dioleyloxy)propyl)-N,N,N-trimethylammonium chlorideand 1,2-bis(oleoyloxy)-3-dimethylaminopropane.
 5. The method of claim 1,wherein said lipid is a member selected from the group consisting ofdimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamineand distearoylphosphatidylethanolamine.
 6. The method of claim 5,wherein said lipid is distearoylphosphatidylcholine.
 7. The method ofclaim 1, wherein said second bilayer stabilizing component ispolyethylene glycol conjugated to a lipid.
 8. The method of claim 1,wherein said second bilayer stabilizing component is polyethylene glycolconjugated to a lipid which is a member selected from the groupconsisting of ceramides, phosphatidylethanolamines,phosphatidylcholines, sphingomyelins and cholesterol.
 9. The method ofclaim 8, wherein said second bilayer stabilizing component is ahydrophilic polymer conjugated to a ceramide having a carbon chainlength between C₈ to C₁₆.
 10. The method of claim 9, wherein said secondbilayer stabilizing component is a hydrophilic polymer conjugated to aceramide having a carbon chain length between C₈ to C₁₆, wherein saidcarbon chain has at least one site of unsaturation.
 11. The method ofclaim 10, wherein said second bilayer stabilizing component ispolyethylene glycol conjugated to a C-₁₄-ceramide lipid.
 12. The methodof claim 11, wherein said second bilayer stabilizing component ispolyethylene glycol conjugated to a C-₁₄-ceramide lipid, saidC-₁₄-ceramide lipid having at least one site of unsaturation.
 13. Themethod of claim 1, wherein said bioactive agent is a member selectedfrom the group consisting of drugs, peptides, proteins, DNA and RNA. 14.The method of claim 1, wherein said faster rate of exchange varies fromminutes to hours.
 15. The method of claim 14, wherein said exchange rateis less than 4 hours.
 16. The method of claim 14, wherein said exchangerate is less than about 1 hour.
 17. The method of claim 1, wherein saidlipid compositions further comprise cholesterol and a second lipid. 18.The method of claim 17, wherein said second lipid is a cationic lipid.19. The method of claim 18, where said cationic lipid is a memberselected from the group consisting of3β-(N-(N′,N′-dimethylaminoethane)carbamoyl)cholesterol,N,N-distearyl-N,N-dimethylammonium bromide,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide, N,N-dioleyl-N,N-dimethylammonium chloride,diheptadecylamidoglycyl spermidine,N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniumchloride, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chlorideand 1,2-bis(oleoyloxy)-3 dimethylaminopropane.
 20. The method of claim1, wherein said second liposome composition comprises a lipid which isdistearoylphosphatidylcholine, a second lipid which is1,2-bis(oleoyloxy)-3-dimethylaminopropane, said bioactive agent is anucleic acid, and said bilayer stabilizing component is a polyethyleneglycol conjugated to a C-₁₄ ceramide lipid.
 21. The method of claim 1,wherein said fluorescent lipid mixing assay is determined by theequation:${\% \quad {Fusion}} = {\frac{\frac{( {F_{(t)} - L_{(t)}} )}{( {F_{T} - L_{T}} )} - \frac{( {F_{O} - L_{O}} )}{( {F_{T} - L_{T}} )}}{\frac{( {M_{(t)} - L_{(t)}} )}{( {M_{T} - L_{T}} )} - \frac{( {F_{O} - L_{O}} )}{( {F_{T} - L_{T}} )}} \times 100}$

wherein: % fusion rate is relative to the % bilayer stabilizingcomponent exchange rate, F_((t)) is fluorescence intensity at time t ofsaid liposome composition; F_(O) is fluorescence intensity at zero timeof said liposome composition; F_(T) is fluorescence intensity in thepresence of a detergent of said liposome composition; M_((t)) isfluorescence intensity at time t for a fused control; M_(T) isfluorescence intensity in the presence of a detergent of said fusedcontrol; L_(O) is fluorescence intensity at zero time of a lightscattering control; L_(T) is fluorescence intensity in the presence of adetergent of said light scattering control; and L_((t)) is fluorescenceintensity at time t for said light scattering control.